Use of mutagenic DNA polymerase for producing random mutations

ABSTRACT

The invention concerns a method for random mutagenesis comprising the replication of a DNA sequence in the presence of an efficient amount of at least a mutase, for example a Pol β, the random mutagenesis rate being at least of the order of 1 mutation for 400 base pairs. The replication product, optionally recombined and amplified, is cloned in an expression vector so as to generate mutated polypeptides which will be selected on the basis of the desired property or properties.

FIELD OF THE INVENTION

The present invention relates to the use of one or more mutagenic DNApolymerases for modifying DNA sequences which are at least partiallycoding. Such a process should make it possible to rapidly obtain a largenumber of random mutants, and therefore mutated polypeptide (for exampleprotein) libraries, and makes it possible to select polypeptides (forexample proteins) of interest according to a predetermined phenotype.This tool is therefore directed in particular toward any experimenterwishing to modify a protein either in the context of a fundamental studyor with a view to a phenotypic improvement for industrial purposes.

BACKGROUND OF THE INVENTION

Various techniques have been developed for promoting in vitromutagenesis on DNA sequences. Among these, mention may be made ofsite-directed mutagenesis and random mutagenesis.

Site-directed mutagenesis is a method which consists in altering thestructure of a protein, in vitro, by simple modification of targetedcodons in the sequence of the DNA. Thus, amino acids can be substitutedat known or supposed active sites of the protein.

Random mutagenesis in vitro consists in introducing, during areplication or recombination step, mutations distributed randomly overthe sequence of a gene or of a gene fragment. The mutations can beintroduced throughout the length of the coding region of a gene, or canbe confined to quite specific DNA segments. Unlike site-directedmutagenesis, a precise knowledge of the structure of the protein is notnecessary to carry out random mutagenesis.

Several methods of random mutagenesis have been developed.

In a first, widely implemented approach, the polymerase chain reaction(PCR) is used under conditions which promote the introduction ofmutations (error-prone PCR). With Taq polymerase, the frequency of basesubstitution can reach 10⁻³, i.e. 1 substitution per 1 000 base pairs(Moore et al. (1996) “Direct evolution of para-nitrobenzyl esterase foraqueous-organic solvant” Nat. Biotech. 14: 458-467). In other cases, afrequency of approximately 1 substitution per 500 bases has been reached(Sweasy et al. (1993) “Detection and characterization of mammalian DNApolymerase β mutants by functional complementation in Escherichia coli”Proc. Natl. Acad. Sci. USA 90: 4626-4630; Diaz et al. (1991)“PCR-mediated chemical mutagenesis of cloned duplex DNAs”. Biotechniques11: 204-211).

However, this technique has several drawbacks. In particular, itrequires a step in which the substrate to be modified is treated withgenotoxic chemical compounds. In addition, it produces too low a rate ofmutagenesis. It is not therefore suitable for the construction of alibrary of mutants and the rapid selection of advantageous mutants.

In another approach, use is made of oligonucleotides (for example 19 or47 base pairs) of random sequence synthesized by a chemical process(degenerate oligonucleotides), which are inserted into a gene,preferably into the region encoding the active part of the enzyme, andused to provide a diversity of proteins. (Horowitz et al. (1986)“Promoters selected from random DNA sequences” Proc. Natl. Acad. Sci.USA 83: 7405-7409; Dube et al. (1989) “Mutants generated by theinsertion of random oligonucleotides into the active site of theβ-lactamase gene” Biochemistry 267: 5703-5707). This procedure requiresthe synthesis, also expensive, of many mutated oligonucleotides.

Yet another approach, based on the use of homologous recombination,similar to the natural process of genetic mixing which takes placeduring evolution, can be used. This method is called shuffling (Stemmer(1994) “DNA shuffling by random fragmentation and reassembly: in vitrorecombination for molecular evolution” Proc. Natl. Acad. Sci. USA 91:10747-10751). It consists in carrying out a PCR on fragments of a geneor fragments of several homologous genes, subsequent to a randomdigestion with DNAse I. The small fragments derived from this digestionserve as primers with respect to one another during the PCR, and lead tothe introduction of random mutations by recombination. This method islaborious and takes place in several steps (digestion of the DNA,recombination by PCR in the presence of “mega-primers”, etc.), whichinvolves difficulties in implementation. It has been used in improvingthe activities of several enzymes such as Green Fluorescent Protein,β-lactamase, and also the arsenate detoxication operon.

The fidelity of several DNA polymerases has been tested in order tomeasure their mutagenic capacity. Among these, the Klenow fragment ofEscherichia coli DNA polymerase I, T4 DNA polymerase, the T7 phage DNApolymerase sequenase, and the Taq DNA polymerase. Among all thesepolymerases, the Taq polymerase exhibits the least fidelity. However,the frequency of mutagenesis remains too low to envisage its use forrandom mutagenic purposes without modification of the reactionconditions or of the polymerase itself (Cadwell et al. (1992)“Randomization of genes by PCR mutagenesis” Cold Spring HarborLaboratory 2: 28-33).

Consequently, despite the techniques developed, there remains, to date,a need for a random mutagenesis technique which is simple to implement,which does not involve the use of genotoxic chemical compounds or ofcomplex steps, or the synthesis of numerous oligonucleotides, and whichgenerates a sufficiently high frequency of random mutations to envisagethe creation of a library of proteins.

DESCRIPTION OF THE INVENTION

After lengthy studies, the Applicant has demonstrated, surprisingly,that it is possible to use a mutase during the replication of a DNA, inorder to obtain a sufficiently high frequency of random mutagenesis.

Document WO 98/23733 describes a method for identifying thermostablemutant polymerases having increased or decreased replication fidelitycompared to a native polymerase. The possibility of using such mutantpolymerases, the fidelity of which is decreased, in a method of randommutagenesis is very briefly mentioned, but no information is given, thedocument focusing on the identification of the mutants and on the use ofthe mutants having increased replication fidelity.

The invention thus relates to a method of random mutagenesis whichcomprises the replication of a DNA sequence in the presence of at leastone mutase in effective amount. The DNA sequence may be a gene fragmentor a complete gene, which can encode a protein, for example an enzyme.

Several mutases can be used, either simultaneously or successively. Themutase(s) used will preferably be thermostable. The mutases which can beused may be native polymerases, i.e. nonmutated polymerases, oroptionally mutated mutases.

The term “mutase” is here intended to mean a DNA polymerase with amutagenic level at least as high as that of DNA polymerase β (Pol β); inother words, a DNA polymerase at least as mutagenic as Pol β. Such amutase will introduce unpaired nucleotides, a source of mutations duringthe replication of the DNA, in a random manner, into a gene or a genefragment.

A suitable mutase can therefore be Pol β, or else Pol ι, Pol η or Pol κ.

DNA Pol β is a small polypeptide of 39 KD. It is an enzyme which ishighly conserved in higher eukaryotes. Its primary function is thoughtto be the “at all costs” repair of damaged DNA, but it also has a rolein the replication of native DNA. DNA Pol β is expressed at a constantlevel during the cell cycle, and exposure of the cells to xenobioticagents, for example radiation, induces its expression.

To date, it has never been used in random mutagenesis. It differs fromthe DNA polymerases conventionally used in mutagenesis by virtue of itsinfidelity during DNA replication, this infidelity being thought to berelated to the absence of associated corrective exonucleated activities(Kunkel T. A. (1985) “The mutational specificity of DNA polymerase-βduring in vitro DNA synthesis” J. Biol. Chem. 260: 5787-5796; Kunkel, T.A. (1986) “Frameshift mutagenesis by eucaryotic DNA polymerases invitro.” J. Biol Chem. 261: 13581-13587). Unlike these other polymerasesused, which exhibit insufficient infidelity to rapidly and easilygenerate random mutations which can result in the creation of mutatedprotein libraries, the degree of infidelity of Pol β is particularlyhigh, and can reach 10⁻².

Various sources of Pol β can be used: Hela cells (cell extracts),chicken, rat and human liver.

A native Pol β or a mutated Pol β can be used.

Whatever the mutase(s) used, the frequency of random mutagenesis is atleast of the order of 1 mutation per 400 base pairs, preferably at leastof the order of 1 mutation per 300 base pairs, more preferably at leastof the order of 1 mutation per 200 base pairs, even more preferentiallyat least of the order of 1 mutation per 100 base pairs, or at least ofthe order of 1 per 50 base pairs.

The mutase(s) used will preferably be in the form of (a) cellextract(s).

The method according to the invention therefore comprises thereplication of a DNA sequence in the presence of an effective amount ofat least one mutase. Those skilled in the art will be able to definethis effective amount. Due to the particular mutagenic properties of themutase(s) used for the replication, copies of the starting DNA sequenceare obtained which are not true copies, and which carry random mutationsin sufficient number to generate a set of mutated polypeptides which canbe used to create a polypeptide library and to select one or morepolypeptides exhibiting one or more desired properties. The polypeptidescan be proteins such as enzymes, of plant or animal, in particularhuman, origin. The desired property(properties) may, for example, beimproved heat resistance, better effectiveness, a more rapid or moretargeted action, binding or improved binding to a receptor, resistanceor improved resistance for certain compounds, etc.

The replication step can be followed by a recombination step. Thisrecombination can be a step of digestion of DNA followed by a PCRamplification step. In addition, during the PCR, the hybridization andpolymerization steps can be combined in a single, very short step. Therecombination can also be a step of digestion of DNA followed by a stepof ligation of the digestion products.

The PCR, when it is used, therefore simply serves to amplify thematerial obtained at the end of the mutagenesis step. Preferably, use isthen made of amplification primers the length of which is such that theyrequire an amplification temperature of at least 70° C., which improvesthe specificity of the PCR.

The replication product, which carries (a) random mutation(s), and whichis optionally recombined and amplified, can then be cloned into anexpression vector in order to generate the mutated polypeptides, whichare isolated. These mutated polypeptides will be grouped together in apolypeptide library, and may be selected as a function of the desiredproperty or properties.

The mutagenic properties of the mutase(s) used may be further increased,in order to further increase the number of mutations generated. Severaltechniques are possible.

Thus, in one embodiment of the method according to the invention,magnesium is substituted with manganese or cobalt in the PCR reaction(Beckman et al. (1985) “On the fidelity of DNA replication: manganesemutagenesis in vitro.” Biochemistry 24: 5810-5817).

Another embodiment of the method according to the invention comprisesthe use of a biased nucleotide pool, i.e. of a pool of nucleotides inwhich one or more nucleotides are favored relative to the others. By wayof example, use may be made of a biased pool in which three of thenatural nucleotides are favored relative to the fourth (Cadwell et al.(1992) “Randomization of genes by PCR mutagenesis.” Cold Spring HarborLaboratory 2: 28-33).

In another embodiment, one or more mutagenic nucleotide analogs areincorporated. Such an analog may be 8-oxoguanine (Zaccolo et al. (1996)“An approach to random mutagenesis of DNA using mixture of triphosphatederivatives of nucleoside analogues” J. Mol. Biol. 255: 589-603).

Use may also be made of another DNA polymerase in combination with themutase(s), whether simultaneously or successively.

Finally, it is possible simply to modify one or more parameters of thereaction medium, such as the pH, the temperature or the saltconcentration, in order to modify the action of the mutase(s). Thepresent application is also directed toward any DNA and any polypeptideor protein obtained, or which can be obtained using a method accordingto the invention. It is in particular directed toward any library ofDNA, of polypeptides or of proteins obtained, or which can be obtainedby collection of DNA, polypeptides or proteins according to theinvention. It is also directed toward any method for identifying acompound exhibiting an activity or property of interest, in particularin the medical, agrofoods or pharmaceutical field, characterized in thatit comprises identifying, in a library according to the invention, acompound exhibiting said particular activity or property.

MORE DETAILED DESCRIPTION OF THE INVENTION

In the following examples, reference is made to the following figures:

FIG. 1 shows very schematically the steps of the method of producingrandom mutations, applied to an 85-mer polynucleotide,

FIGS. 2A and 2B illustrate the results of migrations of the productsthus obtained,

FIG. 3 shows the spectrum of mutations thus engendered by polymerizationof the 85-mer matrix,

FIG. 4 shows schematically the production of random mutations in thelacZ gene carried by a plasmid vector,

FIG. 5 shows very schematically the steps of a method of producingrandom mutations by replication of the lacZ gene encoding the α-peptideof β-galactosidase, carried by the plasmid pUC18, and

FIG. 6 illustrates the distribution of the mutations thus engendered onthe gene encoding the alpha-peptide of the lacZ gene, under standardconditions of replication with Pol β (all the dNTPs at 100 μm)

EXAMPLE 1

In vitro production of random mutations by replication of an 85-merpolynucleotide with DNA Pol β and use of a biased pool ofdeoxynucleotides.

FIG. 1 shows very schematically the various steps of the method, fromthe polynucleotide to the sequencing of the recombinant clones:

Material:

3′ primer: 5′ GATTGAATTCCTCATTATGG 3′ (SEQ ID NO: 1) 5′ primer:5′ TGAATACTGTATGATAATCG 3′ (SEQ ID NO: 2) 85-mer matrix:5′ TGAATACTGTATGATAATCGTGAGGATCCCGCA (SEQ ID NO: 3)TATAAGCTTTTCGATCGCCTGCAGTAACTCCACCAT AATGAGGAATTCAATC 3′

Culture Media

Complete LB medium: 1% tryptone, 0.5% yeast extract, 1% Nacl, H₂O qs 1l. 15 g of agar are added to 1 liter of LB to prepare the LB/agarmedium.

SOC medium: 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10mM MgCl₂, 10 mM MgSO₄, 20 mM glucose.

Bacterial Strain and Vectors

TABLE 4 Strains Genotypes Sources TOP 10 F⁻ mcrA Δ(mrr-hsdRMS-mcrBc)Invitrogen Φ80lacZΔM15 ΔlacX74 recA1 deoR araD139 Δ (ara-leu) 7697 galUgalK rpsL (Str^(R)) endA1 nupG BL21 F⁻ ompT gal (dcm) (lon) hsdS_(B)BioLabs (r_(B) ⁻ m_(B) ⁻) with DE3, has λ prophage carrying the T7 RNApolymerase gene

In Vitro Replication of the Matrix with DNA Pol β Preparation andPurification of DNA Pol β

An overnight culture of the BL21 strain transformed with a plasmidexpressing a Pol β: (His)₆ fusion protein under the control of a T7promoter is diluted to 1/50 in LB medium+50 μg/ml Kanamycin, andincubated at 37° C. with shaking. At OD 0.6-1, 1 mM IPTG is added andthe incubation is continued for 4 hours. The cells are centifuged andthen lysed with lysosyme at 100 μg/ml and 0.1% triton X100. The lysis iscarried out at 30° C. for 30 min. Next, the cells are sonicated and thencentrifuged. The Pol β: (His)₆ fusion is purified on a nickel column andeluted with imidazole using the resin and the buffers of the Novagenpurification kit.

Labeling of the 20-mer primer:

60 ng of the 5′ primer are labeled with 10 units of T₄ polynucleotidekinase (New England BioLabs) for one hour at 37° C. in a buffercontaining 70 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 5 mM dithiotreitol and20 μCi ³²P-γ-ATP. The enzyme is then inactivated by incubating thereaction mixture at 70° C. for 10 minutes.

Hybridization of the labeled primer to the matrix 60 ng of the 5′ primerlabeled with ³²P-γ-ATP are added to 300 ng of the matrix in ahybridization buffer (100 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl₂). Themixture is first denatured at 70° C. for 10 minutes. Hybridization ofthe two oligonucleotides is then obtained by continuing the incubationuntil the temperature of the bath has reached ambient temperature.

Replication of the Matrix

One unit of DNA Pol β is added to 5 ng of the 85-mer matrix hybridizedto 20 ng of 20-mer oligonucleotide labeled with ³²P-γ-ATP, in a reactionmixture containing 25 mM HEPES (pH 8.5), 125 mM NaCl, 5 mM MgCl₂, 200 μMdATP, 200 μM dGTP and 200 μM dTTP. These various mixtures aredistributed into five tubes to which various concentrations of dCTP (0dCTP, 0.2 μM dCTP, 2 μM dCTP, 20 μM dCTP, 200 μM dCTP) are added. Afterincubation for one hour at 37° C., part of the mixture is loaded onto anacrylamide gel (15% acrylamide, 7 M urea, 30% formamide) in order tocontrol the reaction products. The other part is supplemented with dCTPat a final concentration of 200 μM and the reaction is continued for onehour. Finally, the reaction is stopped with the stop buffer (90%formamide/0.1% xylene cyanol/0.1% bromophenol blue/0.1 mM EDTA). Thesamples are then denatured for 10 minutes at 70° C. and loaded onto anacrylamide gel in order to visualize the replication products. Theseproducts are revealed by exposure on an autoradiographic film.

Polymerase Chain Reaction (PCR)

The PCR reaction was carried out by adding 1 μl of the replicationproduct obtained by the action of Pol β to a mixture containing thepolymerization buffer (20 mM Tris HCl (pH 8.8), 10 mM KCl, 10 mM(NH₄)₂SO₄, 0.1% Triton) (New England Biolabs), 1.5 mM MgSO₄, 200 mMdNTP, 20 pmol of each 5′ and 3′ oligonucleotide, and 5 u of highfidelity Vent polymerase (New England Biolabs). These mixtures wereincubated in a BioRad thermocycler according to the following program:(95° C, 5 min)-(95° C. 30 sec.-50° C. 30 sec.-72° C. 30 sec.)×30cycles-(72° C.-5 min.). The PCR products are then loaded onto a 1% TAEagarose gel. After migration and staining of the gel with ethidiumbromide, the 85-mer band corresponding to the product of amplificationof the matrix is visualized by trans-illumination with ultravioletlight.

Cloning

The cloning of the amplification products was carried out using the ZeroBlunt TOPO PCR Cloning Kit from Invitogen. 1 μl of amplification productobtained by PCR is added to 10 ng of the cloning vector pCR-BluntII-TOPO (Invitrogen) contained in the following buffer: 50% glycerol, 50mM Tris-HCl, 1 mM EDTA, 2 mM DTT, 0.1% Triton X-100, 100 μg/ml BSA. Themixture is maintained at ambient temperature for five minutes and thenincubated at 4° C. 2 μl of the cloning reaction are then added to Top 10competent cells (Invitogen), and incubated for 30 minutes at 4° C. Aheat shock is carried out at 42° C. for 30 seconds. The cells are thenagain placed on ice before being incubated at 37° C. for one hour in 250μl of SOC medium. Finally, the transformants are selected on LB/agarmedium containing 50 μg/ml of kanamycin.

Extraction of the Plasmidic DNA

The recombinant plasmids are extracted and purified with the Quia PrepSpin system from Quiagen. The plasmids are eluted in 50 μl of sterilewater and controlled on a gel of 1% agarose in 1×TAE, after stainingwith ethidium bromide and illumination by ultraviolet radiation.

Sequencing

The sequencing reaction is carried out with the SequiTherm EXCEL II DNAsequencing Kits-LC system from Epicentre Technologies. 1 pmol of M13primer labeled with IRD700 (MWG Biotech) and 300 ng of recombinantplasmids are incubated with 1U of Sequitherm EXCEL II DNA polymerase inthe Sequitherm EXCEL II sequencing buffer (Epicentre Technologies). Thismixture was incubated in a PTC-100 thermocycler (MJ Research, Inc.)according to the following program: (95° C. 5 min.)-(95° C. 30 sec.-57°C. 15 sec.-70° C. 1 min.)×30 cycles-(70° C.-10 min.). The reactionproducts are then loaded onto a sequencing gel and analyzed using a LONGREADIR 4200 sequencer (Li-COR).

FIG. 2 shows the result of the migration of the products of replicationwith Pol β. Part A represents the results after replication for one hourwith the various concentrations of dCTP (1=0 μM, 2=0.2 μM, 3=2 μM, 4=20μM, 5=200 μM) and 200 μM of each of the other dNTPs (dATP, dGTP, dTTP).In part B, lanes 6, 7, 8 and 9 represent the products of replication 1,2, 3 and 4, respectively, after complementation of dCTP at aconcentration of 200 μM and continuation of the reaction for one hour.

Table 1 below gives a summary of the production of random mutations byDNA Pol β on an oligonucleotide. It comprises the frequencies of themutations generated by replication with DNA Pol β under standardizedconditions and using a biased pool of deoxyribonucleotides.

TABLE 1 Sequenced Mutation dCTP dATP dGTP dTTP clones frequencies 200μM  200 μM 200 μM 200 μM 26 0.9 × 10⁻² 20 μM  200 μM 200 μM 200 μM 261.2 × 10⁻² 2 μM 200 μM 200 μM 200 μM 27 1.36 × 10⁻²  0.2 μM   200 μM 200μM 200 μM 20 1.7 × 10⁻² 0 μM 200 μM 200 μM 200 μM 18 1.8 × 10⁻²

The experiment under standardized conditions of polymerization with Polβ (the 4 dNTPs are present at a concentration of 200 μM) shows a highmutation frequency, approximately one mutation every 100 nucleotidesreplicated, thus confirming the mutagenic capacity of this polymeraselacking fidelity.

Pol β is capable of completely replicating the 85-mer matrix, even inthe absence of one type of nucleotide out of four. These replicationreactions result in an increase in the rate of mutations which isinversely proportional to the concentration of the fourth nucleotidedCTP (table 1). This confirms the potentialization of the mutageniccapacity of Pol β by the combination of a nucleotide bias in thereaction medium.

Even in the absence of a biased pool of nucleotides, the frequency ofthe mutations observed is 0.9×10⁻², that is to say close to 1 per 100base pairs.

In addition, these mutations are distributed throughout the length ofthe matrix, and in a random manner. The mutagenesis is not thereforeconfined to specific sites.

FIG. 3 represents the spectrum of mutations engendered by thepolymerization of the 85-mer matrix with Pol β under the followingconditions: 200 μM of dATP, dGTP and dTTP, and 0 μM of dCTP.

EXAMPLE 2

In vitro production of random mutations in the lacZ gene carried by aplasmid vector.

The aim of this experiment is to perform SV40 replication of a pBK-CMVmatrix (containing the SV40 origin, the Neo^(r)-Kan^(r) doubleresistance gene and the sequence encoding the α-peptide ofβ-galactosidase allowing α-complementation in Δα-lacZ strains) with Helaextracts and the T antigen, and to add purified rat DNA Pol β during theexperiments. The difference in frequency of mutagenesis induced by theexcess of Pol β is measured using the mutagenesis test described below(white/blue screen).

Bacterial Strains, Plasmids, Restriction Enzymes

The plasmid pBK-CMV (Strategene) was amplified in a dam⁺ bacterium(DH5α) and purified using the Wizard Plus DNA Purification System(Promega). The DpnI enzyme comes from BioLabs.

TABLE 5 Strains Genotypes Sources MC1061Mut S F⁻ araD139 Δ(araleu)7696galE15 T. Kunkel, galk16 ΔlacX74 rpsL (Str^(R)) hsdR2(r_(K) ⁻ USA m_(K)⁺) mcrA mcrB1 [mutS : :Tn10] JM109 F′ traD36 lacI^(q) Δ(lacZ)M15 T.Kunkel, proA + B + /e14⁻ (McrA⁻) Δ(lac-proAB) USA thi gyrA96 (NaI^(r))endA1 hsdR17 (r_(K) ⁻m_(K) ⁺) relA1 supe44 DH5α EndA1 hsdR17 (r_(K)⁻m_(K) ⁺) supE44 thi-1 BioLabs recA1 gyrA (NaI^(r)) relA1 Δ(lacIZYA-argF)U169 deoR (φ80dlacΔ(lacZ)M15)

Preparation of the Replicative Cell Extracts Derived from Hela Cells

The cell extracts were prepared according to the procedure developed byRobert et al. (Robert et al. (1993) “Chromosome and gene analysis”Methods in Molecular Genetics. Adolph, K. W. Ed. 2: 295-313). Hela cellsin suspension are cultivated in 4 liters of RPMI1640 supplemented with9% serum and Pen/Strep5500, harvested in the exponential phase, andcentrifuged at 1 500 rpm for 5 min. at ambient temperature. They arewashed in 200 ml of 1×PBS at 4° C. and centrifuged at 1 500 rpm for 5min. at 4° C. The cells are washed in 25 ml of an isotonic medium at 4°C. (20 mM Hepes-KOH, pH 7.5, 5 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT, 250 mMsucrose) and centrifuged at 4 000 rpm for 10 min. at 4° C. They aretaken up in hypotonic medium at 4° C. without sucrose (20 mM Hepes-KOH,pH 7.5, 5 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT) and then centrifuged. Theyare then lysed in a potter homogenizer after adding 50 μl of aprotininat 1.5 mg/ml, and then centrifuged at 10 000 rpm for 20 min at 4° C. Thesupernatant is then centrifuged at 50 000 rpm for 60 min at 4° C., andthen aliquoted and frozen in liquid nitrogen. The amount of proteinspresent is assessed according to the

Bradford method, using BSA to establish the standard calibration range.

SV40 Replication

This replication step was carried out using the assay developed byRobert et al. The SV40 replication reaction is carried out at 37° C. for6 h in a final reaction volume of 25 μl containing 30 mM HEPES-KOH, pH7.8/7 mM MgCl₂/0.5 mM DDT/200 μM CTP, GTP, UTP/4 mM ATP/100 μM dCTP,dGTP, dTTP/40 mM creatine phosphate/100 μg/ml creatine phosphokinase/100ng pBK-CMV/400 μg of Hela replicative extracts/0.5 μg Large T Antigen towhich are added +/−5 μg (i.e. 1.2 U) of Pol β. The replication reactionis stopped by adding 25 μl of a buffer containing 2 mg/ml PK, 50 mM EDTAand 2% SDS, and incubating at 55° C. for 60 min. The DNA is purified byextraction with phenol, phenol/chloroform/isoamile alcohol, and ether,and then precipitated with 2.5 V 100% ethanol, 0.1 V sodium acetate and1 μg of glycogene, and centrifuged at 13 000 rpm for 30 min. at 4° C.The DNA is then washed with 70% ethanol, centrifuged (13 000 rpm, 15min., 4° C.) and digested with 10 U of DpnI for 2 h in order to removeany pBK-CMV matrix which has not been replicated at least once by theHela extracts. It is finally reprecipitated and taken up in 10 μl ofH₂O.

Analysis of the Mutagenesis

2 μl of replicated plasmid are electroporated into the strainMC1061MutS, which is deficient for the mismatch repair system, in orderto fix the mutations, and then the plasmid is amplified by selecting theelectroporated population with Kanamycin (50 μg/ml) on a total volume of20 ml of LB. The plasmid is extracted using the Wizard Plus DNAPurification System (Promega) and then 100 ng is electroporated into thestrain JM109 allowing α-complementation. A suitable volume of thebacterial population (containing approximately 2 000 to 3 000 clones) isthen inoculated into 7 ml of soft LB containing 1.6 mg of X-Gal, 1.6 mgof IPTG and 350 μg of Kanamycin, and then plated out onto dishes 14 indiameter containing 40 ml of LB/agar with 50 μg/ml of Kanamycin. Thefrequency of mutagenesis present on the replicated plasmid is deducedfrom the number of white colonies compared to the number of totalcolonies present on the dish.

FIG. 4 shows the summary of this production of random mutations.

Table 2 below shows the results of the production of random mutations.

TABLE 2 Colonies examined DNA substrate Total Mutants Mutations × 10⁻³Nonreplicated DNA  28 640 184 6.4 (no (AgT) DNA replicated by 141 390918 6.5 cell extracts without Pol β DNA replicated by 209 762 2 953   14cell extracts with Pol β

It shows that, under physiological conditions, i.e. in the presence ofcell extracts, Pol β is capable of inducing random mutations on amutagenesis target carried by a plasmid replication substrate. Onceagain, it is immediately noted that the frequency of the mutationsinduced is high, in this case 1.4 10⁻², i.e. more than 1 per 100 basepairs.

EXAMPLE 3 Replication of the lacZ Gene by Pol β and Comparison with theResults Obtained by PCR with a taq Polymerase

This example shows that Pol β introduces a frequency of mutagenesis 50times higher than that obtained with taq polymerase by PCR.

Random mutations were produced in vitro by replication of the lacZ geneencoding the α-peptide of β-galactosidase, carried by the plasmid pUC18(invitrogen), with DNA polymerase β, and use of a biased pool ofdeoxynucleotides. FIG. 5 shows very schematically the various steps ofthe method followed, from the replication of the lacZ gene with thepolymerase β to the sequencing of the mutant clones.

Material:

(SEQ ID NO: 4) 5′ primer: 5′ CGCGACTCATGCGACGCATTACGAATTCGAGCTCGGTAC 3′(SEQ ID NO: 5) 3′ primer 5′ CACTCGACGCTGATGCAGTGCACCATATGCGGTGTG 3′

The parts underlined are the sequences complementary to the sequences inthe 5′ and 3′ positions flanking the lacZ gene carried by the plasmidpUC18.

Culture Media

The composition of the Medium is Detailed in Example 1.

Bacterial Strain

The genotype and the origin of the strain Top 10 are described inexample 1.

In Vitro Replication of the lacZ Gene with DNA Polymerase β

1 μg of plasmid pUC18 is added to 10 pmol of the 5′ and 3′ primers in 15μl of replication buffer (50 mM Tris-HC1, pH 8.8, 10 mM MgCl₂, 100 mMKCl, 0.4 mg/ml BSA, 1 mM DTT, 10% glycerol, Trevigen). The mixture isdenatured for 5 min. at 90° C. and then hybridized for 2 min. at 55° C.,before being conserved at 4° C. A solution of 15 μl of the same buffercontaining 4 units of polymerase β (Trevigen), 0.5 mM Mn2+ and 100 μM ofeach of the 4 natural deoxynucleotides is then added to the 15 μl ofhybridization mixture. The reaction of replication by polymerase β isthen carried out at 37° C. for one hour. In other polymerizationreactions, the pool of the 4 deoxynucleotides is biased by a lowerconcentration of one of the 4 deoxynucleotides compared to the threeothers. The replication products are then extracted withphenol-chloroform.

Selective PCR

The selective PCR reaction is carried out by adding 1 μl of thereplication product (diluted to 1/10), obtained by the action of thepolymerase β, to a mixture containing the PCR buffer (20 mM Tris HCl pH8.4, 50 mM KCl) (Life Technologies), 1.5 mM MgCl₂, 10 pmol of the 5′ and3′ primers, 200 μM of the 4 dNTPs and 1.25 U PLATINUM Taq DNA polymerase(Life Technologies). This mixture is incubated in a thermocycler(Eppendorf) according to the following program: (95° C., 5 min.)-(94°C., 15 sec.-55° C., 30 sec.-72° C., 30 sec.)-(94° C., 15 sec.-72° C., 30sec.)+30 cycles-(72° C., 10 min.). This program makes it possible tospecifically amplify the DNA fragments synthesized by the polymerase β.

The PCR products are then extracted with phenol-chloroform, precipitatedwith ethanol, and recovered in TE (10 mM Tris HCl pH 8, 1 mM EDTA).

Cloning of the PCR Products

Before being cloned into pUC18, the PCR products are digested with 20 Uof NdeI (BioLabs) and 10 U BamH1 (Q-BIOgene) in a digestion buffercontaining (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT and 100μg/ml BSA) for one hour at 37° C. The digestion product is then purifiedwith the GenClean2 kit (BIO 101) after migration on agarose gel andexcision of the band containing the PCR product. The PCR product is thencloned into pUC18 (predigested with NdeI and BamH1) subsequent to aligation (16° C. overnight) with 40 U of T4 DNA ligase (BioLabs) in abuffer containing (50 mM Tris HCl pH 7.5, 10 MM MgCl₂, 10 mM DTT, 1 mMATP and 25 μg/ml BSA). The ligation product is then precipitated withethanol and taken up in 5 μl of TE (10 mM Tris HC1 pH 8, 1 mM EDTA). 2μl of the ligation reaction are then added to the Top 10 competent cells(Invitogen) and incubated for 30 minutes at 4° C. A heat shock iscarried out at 42° C. for 30 seconds. The cells are then again placed inice before being incubated at 37° C. for one hour in 250 μl of SOCmedium. Finally, the transformants are selected on LB/agar mediumcontaining 100 μg/ml of ampicillin and 60 μg/ml of X-Gal(5-bromo-4-chloro-3-indolxyl-beta-D-galactopyranoside, Euromedex). Thefrequency of mutants is deduced from the number of white coloniescompared to the number of total colonies present on the dish.

FIG. 5 shows the summary of this production of random mutations.

FIG. 6 illustrates the distribution of the mutations thus engendered onthe gene encoding the alpha-peptide of the lacZ gene, under the standardconditions for replication with Pol β (all the dNTP at 100 μM).

Table 3 below shows a summary of production of random mutations.

TABLE 3 Mutation Clones frequency screened Mutants (%) Mutations/kbReplication by Taq 2 908  14 0.48 ND (100 μM dNTPS) Replication by Pol β  764 187 24.48 17.09 (100 μM dNTPs) Replication by Pol 1 749 730 41.7418.47 β (100 μM dATP, 100 μM dGTP, 100 μM dCTP, 20 μM dTTP) Replicationby Pol 1 734 1 078   62.17 23.6 β (100 μM dGTP, 100 μM dCTP, 100 μMdTTP, 20 μM dATP)

This summary shows that, under normal conditions, i.e. when thereplication with polymerase β is carried out with 100 μM of each of thefour dNTPs, the polymerase β is capable of producing random mutations onthe gene encoding β-galactosidase. This rate of mutation (24.48%),compared to that produced by PCR amplification using the taq polymerase(Life Technologies) (0.48%), is 50 times higher. This very high rate ofmutation produced by polymerase β makes it possible to screen a smallernumber of mutants with the aim of isolating a mutant of a protein whoseactivity is enhanced or weakened. This frequency of mutants is evenhigher when the concentration of one of the four dNTPS is modified. Forexample, when the dTTP or the dATP is used separately in two independentreactions at a concentration of 20 μM, the frequency of mutants obtainedis 41.7% and 62.17% respectively. This increase in frequency of mutantsmakes it possible to reduce even further the number of mutants to bescreened.

The results of these various experiments demonstrate the relevance ofusing this enzyme, which is in particular native, for the purpose ofcreating random mutants, and make it possible to envision designing atool based on the use of Pol β or of cell extracts/Pol β for the purposeof modifying any sequence carried by a plasmid vector.

1. A method of random mutagenesis comprising steps of: (i) replicating aDNA sequence with an effective amount of at least one mutase in order togenerate random mutations within the replicated DNA sequence, whereinthe at least one mutase is selected from the group consisting of DNApolymerases beta, iota, eta and kappa; (ii) amplifying by PCR thereplicated DNA using a DNA polymerase other than those recited in step(i); (iii) cloning the amplified DNA sequence into an expression vector;and (iv) generating a library of mutated polypeptides.
 2. The method ofclaim 1, wherein the at least one mutase is DNA polymerase beta.
 3. Themethod of claim 1, wherein the at least one mutase is used in the formof a cell extract.
 4. The method of claim 1, wherein several mutasesselected from the group consisting of DNA polymerases beta, iota, etaand kappa are used simultaneously in the DNA replication in step (i). 5.The method of claim 1, wherein several mutases selected from the groupconsisting of DNA polymerases beta, iota, eta and kappa are usedsuccessively in the DNA replication in step (i).
 6. The method of claim1, wherein random mutations within the replicated DNA sequence aregenerated with a frequency at least of the order of 1 mutation per 400base pairs.
 7. The method of claim 6, wherein the frequency is at leastof the order of 1 mutation per 300 base pairs.
 8. The method of claim 7,wherein the frequency is at least of the order of 1 mutation per 200base pairs.
 9. The method of claim 8, wherein the frequency is at leastof the order of 1 mutation per 100 base pairs.
 10. The method of claim9, wherein the frequency is at least of the order of 1 mutation per 50base pairs.
 11. The method of claim 9, wherein the DNA replication instep (i) is followed by a recombination step.
 12. The method of claim 1,wherein nucleotides selected from the group consisting of a biasednucleotide pool, one mutagenic nucleotide analog, several mutagenicnucleotide analogs and/or any combination thereof are used in the DNAreplication in step (i).
 13. The method of claim 1, wherein an elementselected from the group consisting of manganese and cobalt is usedduring the PCR amplification of step (ii).
 14. The method of claim 1,wherein cloning the amplified DNA sequence into an expression vectorcomprises steps of: synthesizing the mutated polypeptides; and isolatingthe mutated polypeptides synthesized.
 15. The method of claim 14,wherein the amplified DNA sequence is recombined before being cloned.16. The method of claim 14, wherein mutated polypeptides exhibiting adesired property or desired properties are selected.
 17. The method ofclaim 1, wherein the DNA replication in step (i) involves replicating aDNA sequence carried by a plasmid.
 18. The method of claim 1, whereinthe DNA replication in step (i) is stopped with a stop buffer prior tocommencement of the PCR amplification in step (ii).
 19. The method ofclaim 1, wherein the DNA replication in step (i) is carried out atemperature less than 70°C., and the PCR amplification in step (ii) iscarried out at a temperature greater than 70°C.
 20. The method of claim1, wherein the polymerase used for the DNA replication in step (i) isstable only at a temperature less than 70°C., and the polymerase usedfor the PCR amplification in step (ii) is stable at a temperaturegreater than 70°C.
 21. A method of random mutagenesis comprising stepsof: (i) replicating a DNA sequence with an effective amount of at leastone mutase to generate random mutations within the replicated DNAsequence, wherein the at least one mutase is selected from the groupconsisting of DNA polymerases iota, eta and kappa; (ii) amplifying byPCR the replicated DNA using a DNA polymerase other than those recitedin step (i) (iii) cloning the amplified DNA sequence into an expressionvector; and (iv) generating a library of mutated polypeptides.